Fig 1.
Characterization of Ixodes RNAi factors.
(A) Phylogenetic analysis of RdRP and AGO genes. The sequences of AGO and RdRP homologs from representative organisms (Ixodes, C. elegans, Arabidopsis thaliana, Neurospora crassa and Schizosaccharomyces pombe for RdRP; Ixodes, Drosophila, C. elegans and human for Ago/PIWI) were aligned using MUSCLE. Gene names are surrounded by colored rectangles depending on the species (Fungi: gray, A. thaliana: green, C. elegans: orange, Drosophila: blue, human: yellow, Ixodes: dark red). (B) Protein domains found in I. scapularis RdRP, PIWI and AGO genes. The domains were identified using the CD-search tool (NIH/NLM/NCBI). (C) Expression of tick RNAi factors analyzed by RNAseq. The dataset of ISE6 total RNAseq from the control sample (dsGFP transfection) was used to determine the expression levels of the indicated genes. Expression levels are shown as TPM and the averages and standard deviations are shown in the bar chart (n = 3). (D) Subcellular localization of tick AGOs and RdRPs in HEK293T cells. HEK293T cells were transfected with the indicated plasmids and fixed. The cells were stained with Hoechst and observed by confocal microscopy. The bars indicate 50um.
Fig 2.
Characterization of endogenous sRNA populations in ISE6 cells.
(A) Genomic origins of sRNA populations in ISE6 cells. The fractions of sRNAs derived from miRNAs (blue), RNAP III transcribed genes (pink), rRNAs (orange), snoRNAs and mRNAs (yellow) and repetitive sequences (green) are shown. sRNA reads whose genomic loci lack a gene annotation are shown as “No annotation” (light green). The percentage of reads falling within each category was calculated by dividing the number of reads in the category by the number of reads mapping to the ISE6 genomic sequence. (B) Normalized read count (RPM) of sense and antisense reads that mapped to the reference sequences of miRNAs, RNAP III transcripts, mRNAs and repeats in the indicated knockdown libraries are shown in blue and red, respectively. Negative values were given to antisense read counts. (C) Example of sRNAs in the “RNAP III” category. A UCSC screenshot of the locus of the RNAP III transcribed gene, RNase MRP RNA gene is shown, with y-axis representing the normalized read counts. On the track of “standard sRNAseq library”, reads corresponding to an abundant 22nt species are mapped on the antisense strand of the RNase MRP RNA locus, and there are reads throughout both sense and antisense strands of this locus. To analyze chemical structures at their 3’ nucleotides, we generated sRNA libraries after oxidizing RNA samples to enrich for sRNA species containing chemical modifications at the 2’-position. Nucleotides with free 2’-, 3’-OH groups react with periodate to form dialdehydes, which are not compatible with the 3’-linker ligation. Therefore, species with 2’-O-me modification will be overrepresented in the oxidized library. sRNAs from RNAP III-transcribed genes tended to be depleted in the oxidized library suggestive of the presence of vicinal hydroxyl groups at the 3’-nucleotide of the sRNAs. The phosphorylation status of the 5’-end could also be analyzed by taking advantage of the 5’-mono-P specificity of T4 RNA ligase, which is used for library construction. For standard sRNA library construction, the 5’-linker ligation step strongly enriches for 5’-mono-P species. For the “5’-tri-P” library, sRNAs with 5’-di-P or 5’-tri-P groups were enriched by the removal of sRNAs with 5’-mono-P and 5’-OH groups by terminator exonuclease (See Materials and Methods). No enrichment was seen with the sRNA species from RNAP III-dependent loci in this library, suggesting that the sRNAs were 5’-mono-phosphorylated. (D) 5’- and 3’-states of a piRNA (TE- family-423-A), an antisense sRNA from a RNAP III dependent gene (tRNA ISCW004624) and a miRNA (miR-8) were verified by Northern blotting. The removal of phosphate groups at the 5’-end causes a delay in the migration on the gel. The alkaline treatment removes the oxidized RNA with dialdehydes at the 3’ ends, resulting in a faster migration of the RNA on the gel (ß-elimination). (E, F) Antisense sRNAs are produced from the RNAP III-dependent gene RNase P in Asian longhorned ticks. sRNA mapping at the RNase P locus is shown (E), with y-axis representing the normalized read counts. The sRNA library was made using RNA samples extracted from purified extracellular vesicles in saliva. The size distribution of sRNAs mapping to the representative RNAP III-dependent genes (RNase P, RNase MRP and SRP RNA) are shown. RPM normalized sense and antisense read counts are shown in blue or red bars, respectively. Note that strong peaks at 22nt were observed in saliva and tick animals at different developmental stages at least on the antisense strand, indicating the conservation of an sRNA production mechanism similar to those observed in ISE6 cells (Also see S6 Fig).
Fig 3.
(A) UpSet plot [47] of sRNA factor dependencies for sRNA-producing coding genes. The genes that had >35 RPM on average in the ten sRNA libraries were considered. If the sRNA reads from the locus were reduced by >40% in the knockdown library, the sRNA was judged as “dependent” on the factor that was knocked down. The number of dependent genes in each group is shown. Note that many genes were dependent on the Aub-AGO3 (blue) and RdRP3-Ago-16 (pink) combinations. (B) Read counts from protein-coding sequences in the control GFP-KD library are shown in the bar chart. sRNA reads derived from the annotated coding regions that had >35 RPM on average in the ten knockdown sRNA libraries are shown. If sRNA reads were reduced by >40% upon knockdown of one of the indicated sRNA factors, the gene was judged dependent on the factors. sRNA read size distributions of a representative locus in each group are shown in the bar charts below. If sRNA reads were reduced by >40% by knocking down any of the indicated sRNA factors in the group, bars are colored according to the following color-code (Blue: Aub-AGO3-1, or AGO3-2; Pink: RdRP3 or Ago-16; Green: RdRP1 or Ago-30).
Fig 4.
(A) UpSet plot [47] of sRNA factor dependencies for sRNA-producing repeats. The repeats that had >800 RPM on average in the ten sRNA libraries were considered. If the sRNA reads from the locus were reduced by >40% in the knockdown library, the sRNA was judged as “dependent” on the factor that was knocked down. The number of dependent genes in each group is shown. Note that many genes were dependent on the Aub-AGO3 (blue), RdRP3-Ago-16 (pink) and RdRP1-Ago-30 (green) combinations. (B) Read counts of repeat-associated sRNAs in the control GFP-KD library are shown in the bar chart. Repeats that had >800 RPM on average in the ten knockdown sRNA libraries are shown. If sRNA reads were reduced by >40% by knocking down any of the indicated sRNA factors in the group, bars are colored according to the following color-code (Blue: Aub-AGO3-1, or AGO3-2; Pink: RdRP3 or Ago-16; Green: RdRP1 or Ago-30). sRNA read size distributions of a representative repeat in each group are shown in the bar charts below.
Fig 5.
Roles for the RdRP-dependent pathways in gene regulation.
(A-C) MA-plots of pairwise comparisons between the control (dsGFP) and the knockdown sample (n = 3). Red dots indicate genes differentially expressed in the knockdown samples (adjusted p <0.05) and RNAi-related gene names are indicated if they are differentially expressed and highlighted by green circles. The point representing Dsor1 was highlighted by a blue circle. (D-F) GO-term analysis of misregulated genes revealed in the above analysis. Results of biological process analysis are shown. The size and color represent the number of genes in the GO category and the significance of enrichment, respectively, as indicated in the legend. Results of molecular function and cellular component groups analyses are shown in S1 File.
Fig 6.
Dsor1 is a target of the RdRP1-dependent sRNA pathway.
(A) Sreenshot of UCSC genome browser of the locus of the RdRP1-regulated gene Dsor1 (ISCI005428). The original gene structure annotation in the Vectorbase (ISCI005428) lacks the UTRs but reads corresponding to the extending transcript beyond the coding region are visible on the RNAseq mapping data. Within its 3’UTR, there is a high peak of sRNAs ("sRNA peak"). The Y-axis of the mRNA-seq and sRNA-seq mapping tracks means the normalized read counts. (B) sRNA reads mapping to the sense or antisense strands of Dsor1 locus was quantified. A dramatic reduction of sRNAs mapping to both sense and antisense strands was seen upon RdRP1 knockdown. (C) Expression levels of Dsor1 mRNA as analyzed by the salmon pipeline. The averages and standard deviations of TPM values are shown (n = 3). The results were verified by qPCR (Right panel, n = 4). (D) The Dsor1 3’UTR was cloned in the pmirGLO/Fer-Luc2/Act-hRluc vector [54], and ISE6 cells that were soaked with the indicated dsRNA were transfected with the sensor plasmid. The ratio between firefly luc and Rluc was normalized to that of the empty sensor, and the means and standard deviations are shown in the chart (n = 7). The experiments were performed twice on different days and the results from the two experiments were combined. Student’s t-test was used to calculate p-values.
Fig 7.
sRNAs from persistently infecting viruses.
(A) sRNA reads from non-treated ISE6 cells were mapped to the 5 viral genomes that were known to be present in ISE6 cultures. As a representative virus, LC094426 (iflaviridae) is shown. Read density was calculated at each base and normalized for the number of reads mapping to the ISE6 genome. (B) Size distribution of viral sRNAs. Viral sRNAs of each length were counted and normalized to the number of reads mapping to the ISE6 genome (reads per million genome mapping reads). The result of the non-treated control cells is shown. (C) Changes in the sRNA (Upper, n = 1) and the viral transcript (Lower, n = 3) abundances in ISE6 cells after knocking down AGO or RdRP. sRNA libraries and total RNAseq libraries were analyzed to quantify the abundances of viral sRNAs and viral transcripts in the indicated libraries. Fold change values were calculated based on reads per million genome mapping reads (for sRNAs) and TPM (for viral transcripts). The averages and the standard deviations are shown. The p-values were calculated by comparing each group with the control (dsGFP) group and the asterisks indicate the significance (*: p<0.05, **: p<0.01; n = 3, t-test). (D) Changes in the viral transcript level upon RdRP1-Dsor1 double knockdown. Cells treated with the indicated dsRNAs were used. The amount of dsRNA was adjusted by adding lacZ control dsRNA so that each sample is treated by the same amount of dsRNA. qPCR primers detecting the indicated viruses were used and the values were normalized by actin and expressed as fold change relative to the level in the lacZ dsRNA control sample. T-tests were performed in all combinations and the number of asterisks indicate the significance (n.s.: p>0.05, *: p<0.05, **: p<0.01; n = 12). (E) Working hypothesis. The tick has various sRNA pathways, some of which control viral transcripts by vsiRNAs and some others control antiviral response by controlling Dsor1 expression by RdRP1-dependent endogenous sRNAs.